Methods and compositions for controlling animal parastites



United States Patent METHODS AND COMPOSITIONS FOR CON- TROLLING ANIMALPARASITES Konrad Bernhauer and Wilhelm Friedrich, Aschalfenburg,

Germany, assignors to Firma Aschatrenburger ZellstotfwerkeAktiengesellschaft, Redenfelden, Upper Bavaria, Germany No Drawing.Application December 17, 1953 Serial No. 398,854

Claims priority, application Germany December 20, 1952 3 Claims. (Cl.167-81) vitamins of the B group from the various natural products is theproduction of concentrates. This task is accomplished for example in themanner that an aqueous extract is produced from the starting material,and this is treated with an absorbing agent such as activated f carbonand the adsorbate is eluted with aqueous alcohol. a

After the concentrating of the eluate in vacuum to a small volume thereis obtained a highly active concentrate of the vitamins of the B group.The present invention refers to the further treatment of suchconcentrates.

Concentrates of this type can now be further worked a up in variousways, for example the aqueous solution of the anti-anemia factor isextracted with phenol. According to a known process, the followingmethod of treating is utilized: the vitamin B -containing solution at apH value of 1.0 is extracted with an alkylated phenol, preferablyo-amylphenol.

The further purification of this concentrate depends upon thetransferring of the vitamin B from the organic phase into the aqueousphase. This has been done either by the addition of a large amount ofliquid of the type in which the vitamin B is not soluble and at the sametime is not miscible with water, i. e. ethyl ether, petroleum ether,chloroform, etc., or by steam distillation.

The above mentioned aqueous concentrates produceable by theconcentration of eluates can also be worked up in this manner so thatthe vitamin B after addition of an inorganic salt, especially ammoniumsulfate, can be obtained by extraction with alcohol. It has been found,however, that the saturation of the concentrates of the vitamin B groupwith inorganic salts such as ammonium sulfate results in thatpractically the entire vitamin B activity is separated contained in theprecipitate with or without the application of a filter aid. In anycase, there is obtained an enriching of the vitamins of the B groupwhich further need a very basic purification which is based upon asystematic separation of impurities, be-

ing obtained in the usual manner from a raw product containing the Bgroup vitamins, i. e. liver extract or Streplomyces griseus.

It is still another object of the present invention to provide a processof transferring the vitamins of the B group, obtained in an organicphase from the dry or aqueous vitamin B concentrate which in turn hasbeen obtained from a raw product, into an aqueous phase.

It is another object of the present invention to provide a process ofprecipitating the vitamins of the B group from the aqueous phase.

Other objects and advantages of the present invention will be apparentfrom a further reading of the specification and of the appended claims.

With the above objects in view, the process of treating a solid dryconcentrate or an aqueous concentrate containing the vitamins of the Bgroup and obtained in the customary manner, mainly comprises the stepsof treating a concentrate containing vitamins of the B group with anorganic solvent mixture comprising a phenolic substance dissolved in aliquid organic solvent in which the vitamins of the B group areinsoluble, whereby the vitamins of the B group are dissolved in thesolvent mixture, and recovering the thus obtained organic solution ofthe vitamins of the B group.

According to a preferred embodiment of the present invention, an aqueousconcentrate is treated with a halogenated phenol dissolved in ahydrocarbon, a halogenated hydrocarbon or carbon disulfide for theextraction of the vitamin B group. The dry vitamin B concentrate may betreated with any phenol dissolved in a solvent therefor, which solventis not of itself a solvent for the vitamins of the B group, i. e. ahydrocarbon, a halogenated hydrocarbon or carbon disulfide. It is to beunderstood that mixtures of such phenols and/or mixtures of the" solventtherefor may be used instead of just the individual substances.

According to another preferred embodiment of the present invention, thehalogenated phenols which are utilized with a hydrocarbon, halogenatedhydrocarbon or carbon disufide solvent therefor, for the extraction offore it is possible to obtain crystallization of the vitamins It is afurther object of the present invention to provide a process ofextracting the vitamins of the B group from either a dry concentrate oran aqueous concentrate containing the vitamins, such concentrate bethevitamins of the B group, particularly from an aqueous concentrate,contain the halogen group or groups preferably in the meta, and mostpreferably in the para position.

The process of the present invention may be carried out either on thecountercurrent principle or by extraction with fractions.

The above described treatment of the dry or aqueous concentratecontaining the vitamin 13 group results in an organic solutioncontaining these vitamins. The present invention then contemplates theremoval of the vitamins of the 13 group from this organic phase and thisis preferably accomplished by adding a small amount of anoxygen-containing polar organic substance to the organic solution,whereby the solubility of the .vitamins of the B group in the organicmedium is either partially, and in fact generally completely, removed.The oxygencontaining polar substance which is utilized may either be ofthe type in which the vitamins of the B group are soluble i. e. analcohol or the like, or a substance in which the vitamins of the B groupare not soluble, i. e. a ketone, ether, ester or the like. In any case,after the addition of the oxygen-containing polar organic substance tothe organic phase, the vitamins of the B group may be extracted from theorganic phase by means of water. This may be done either by adding thewater directly to the mixture or by first isolating the vitamins of theB group, precipitated from the organic phase by an oxygencontainingpolar organic substance in which the vitamins of the 13 group are notsoluble, and then treating with Water.

The term phenolic substance as used throughout the specification andclaims is meant to include phenol, alkyl, aryl and other derivativesthereof and more particularly the halogenated phenols. As shall be moreclearly explained, derivatives having a substituent in the orthoposition, particularly where the substituent is of large size, are notdesirable and are therefore not meant to be in cluded within the termphenolic substances. The term solvent agent as used throughout thespecification and claims is meant to include those liquid organicsubstances, namely hydrocarbons, halogenated hydrocarbons and carbondisulfide in which the phenolic substance is solbe such liquid which cansuitably dissolve the halogenated phenols. The very surprising andimportant discovery of the superiority of the halogenated phenols overnonhalogenated phenols in treating aqueous concentrates may be seen fromthe following table, the best results being obtained if the halogenatedphenols has the halogen situated in meta or para position, i. e.m-chorophenol or p-chlorophenol.

Although the scope of the present invention is not meant to be limitedthereby, theoretical discussion will be given throughout in the hopethat it will help others to better understand the process of theinvention.

TABLE 1 Phenol content in trichloroethylene in which the distributionPosition coefiicient for B is about 1 in Mol/Ltr In percent Bun No.Phenol derivative Aqueous] Aqueous/ of the of the Volume Proportionorganic Volume Proportion organic Halogens Methyls phase phase phenol 0.95 2. 05 3. 3 8. 93 19. 3 31.0 o-chlorophenol ortho. 2. 24 28. 8m-chlorophenol meta 0. 42 0. 6 0.76 5. 4 7. 7 9. 8 p-chlorophenol para0. 6 0.8 1. 12 7. 7 10.3 14. 4 2, fi-dichlorophcnoL... {g2 1. 67 27. 22, G-dichlorophenol. {gigg }in saturated solution no extraetability ofvitamin B12 2, 4-dich1orophcnol.... {gg g?: 1. 76 l 21. 5 I

ortho.. 2, 4, 6-trich1orophenol ortho.. in saturated solution noextractability of vitamin Bu para... ortho 2, 4, GtribrornophenoL ortho.in saturated solution no extractability of vitamin B17 para5-chloro-2-cresol.

orth0... orthom.

0. 64 O. 64 0 4 9. 1 in saitiarzated solution no extractability ofvitamin B uble but in which the vitamins of the B group are insoluble.The term solvent mixture is meant to comprehend a mixture of suchsolvent agents and phenolic substance in which the vitamins of the Bgroup are soluble. The term concentrate, unless specifically modified,is meant to include either a dry concentrate or an aqueous concentrateobtained in the usual manner from a raw product containing the vitaminsof the B group, i. e. liver extract.

Contrary to the prior known processes, the present invention comprises:

A. The extraction of the vitamins of the B group from an aqueousconcentrate with halogenated phenols dissolved in liquid hydrocarbons,halogenated hydrocarbons or in carbon disulfide;

B. The extraction of the dry powdery concentrate which contains thevitamins of the B group with a phenolcontaining solvent mixture;

C. The transfer of the B vitamins from the phenolcontaining organicphase into an aqueous phase; and

D. The precipitation of the vitamins of the B group from the aqueoussolution by small amounts of suitable phenols.

The individual process steps of the present invention will be more fullydiscussed further in the specification.

It has been found that the above mentioned proceeding according to theknown processes for the extraction of the vitamins of the 3 group froman aqueous concentrate containing impurities can be considerablyimproved it there is used halogenated phenols dissolved in certainsolvents which are not very miscible with water, i. e. hydrocarbons orhalogenated hydrocarbons or carbon disulfide, this serving to extractthe B vitamins from the aqueous concentrate. The solvent to be suitableshould ability for the vitamins of the B group.

From Table 1 it may be seen that in the ortho position, mono-halogenatedphenols have a weak dissolving ability, in ortho position di-halogenatedphenols have no dissolving ability for vitamins of the B group; further,that in para position halogenated phenols in general have a betterdissolving ability for vitamins of the B group than non-halogenatedphenols; as well as that a halogen in meta position very greatlyincreases the dissolving ability of the phenol for the vitamins of the Bgroup. Halogenating of cresol in general considerably increases thedissolving ability for vitamins of the B group, above all in consideringthe molar concentration; an exception occurs with 6-chloro-2-cresol,which has no dissolving This pertains only to solutions of phenols insolvents mentioned above but not to phenols themselves.

The superiority of the halogenated phenols when extracting from anaqueous concentrate, particularly as compared to phenol, is especiallyapparent upon extraction with smal volumes of the organic phase. It isthus necessary, for example by an extraction with /5 volume ofextracting agents, i. e. with trichloro-ethylene, plus a phenolicsubstance, to utilize 19.3% phenol but only 9.1% 6-chloro-3-cresol,10.3% p-chlorophenol or 7.7%

m-chlorophenol, if the distribution coefficient for the vitamins of the13 group has a value of 1.

A still greater superiority of the halogenated phenol, as compared forexample to phenol, in extracting from aqueous concentrates, arises bythe extraction with volume of extracting agent. It has been noticed thatfor example only 14.4% of p-chlorophenol in trichloro-ethylone isnecessary as compared to more than double the amount of phenol (31.0%).If the calculation is made in relation to the molar concentration, it isshown, as in Table I, that the superiority of the mentioned halogenatedphenol is still more clearly apparent. I

The rapid increase in the need for the phenol with the lowering of thevolume of extracting agents may be traced back to the relatively highsolubility of the phenol in water. The phenol divided itself between thewater and the phenol carrier, i. e. the trichloroethylene, and thehigher the relative volume of the aqueous phase, the more phenol is lostduring the extraction. The phenomenon occurs with halogenated phenolsonly to a very slight extent, which is traceable back to the slightsolubility of the substances in water. Thus, the need for 6-chloro-3-cresol is practically not increased at all with lowering of the volumeof extracting agents, as may be seen in Table l.

The concentrations of halogenated phenol in the organic phase to be usedin this extraction process are evident from the above table, though theamounts are given as exemplary only.

The extraction of the aqueous phase with the above named solventmixtures can be carried out in separatory funnels or, on thecountercurrent principle, in batteries of separatory funnels, or insolvent-centrifugal-extractors.

In the following description of the present invention for thepurification of concentrates of the vitamin B group which results in aconsiderable improvement of the known products, a dry product serves asstarting material, the dry product containing vitamins of the B groupand are obtained by one of the following two methods:

(1) A solution reduced to a small volume and containing the B vitaminsis saturated i. c. with ammonium sulfate and at the same time extractedwith a higher alcohol, i. e. n-butanol, amyl-alcohol or benzylalcohol.The extract is evaporated to dryness under vacuum and the dry product ispulverized.

(2) The same type of solution as in 1 above is likewise saturated withammonium sulfate. The thus resulting precipitate which contains thevitamins of the B group is, as such, or after addition of a filter aidsuch as diatomaceous earth, separated and either immediately or invacuum dried or by mixing with roasted sodium sulphate or gypsum or thelike dehydrated and the dry product pulverized. 4

The process according to the present invention thus also concerns theextraction of a dry product containing vitamins of the B group. Thedried powder containing vitamin B obtained according to one or both ofthe described methods is extracted with a solvent mixture. This consistsof phenol or a halogenated phenol and a suitable solvent therefor whichis not miscible with water and in which alone the vitamins of the Bgroup cannot be dissolved, for example a low hydrocarbon or a lowhalogenated hydrocarbon or carbon disulfide. Solvents of this type serveonly as phenol carrier and are so designated in the remainder of thespecification. By this extraction the vitamins of the B group go intosolution while the entire preponderant amount of the inorganic salt asWell as a considerable amount of organic impurities remain behindundissolved.

The process can, however, also be carried out in such manner that theextraction of the dry powder containing the vitamins of the B group isby fractional percolation with a series of solvents and/or solventmixtures. For this purpose the dry material is stirred with acetone andin mushy slimy-like state it is filled in the percolator. It is then, ifnecessary under pressure, or at increased temperature thoroughly washedwith acetone whereby first yellow-colored impurities go into solution.It is then washed with one of the above named phenol carriers or solventagents, first without the addition of a phenol (or with another suitablesolvent in which the vitamins of the B group are not soluble) wherebyfurther impurities are dissolved.

The resulting percolate up to this point is practically free of vitaminsof the B group and can therefore be disregarded. Finally, the product ispercolated with a mixture of a phenol carrier with a phenol (a solventmixture). The content in this stage of the applied percolating liquid inphenolic substances should be more than 5%. Although, for exampletrichloroethylene has a pronounced ability for extracting the vitaminsof the B group from water, only after the addition of about l0-15phenol, it is possible according to the present invention, by the use ofa solid extraction material (a solid concentrate) to obtain an excellentyield with only a 510% phenol solution. Already with a content of 5%phenol or a halogenated phenol, i. e. iu'trichloroethylene, there may beseen a weak dissolving action with respect to the vitamins of the Bgroup, while it is practically impossible with'a similar mixture toextract the yitamins of the B group from an aqueous solution.

With the described process there is the possibility on the one hand bythe pro-percolation by means of solvents in which the B vitamins are notsoluble, to remove considerable amounts of impurities, and on the otherhand there remains a great deal of impurities in the percolator afterthe main percolation. The resulting percolate which is ready forfurfl-ler treatment and. which contains the vitamins of the B group hasa deep red color and can with advantage be further treated to obtaincrystals of the B vitamin group.

The process of the present invention has the advantage that it ispossible to utilize a relatively low amount of solvent and a very muchreduced amount of phenolic substances. This results on the one hand inthe advantage that it is possible to work in small preparation vessels,which, therefore, results in a greater production capacity, and on theother hand in that the preparation is facilitated since substantiallysmaller amounts of phenols need to be removed in the further running ofthe treatment. It is self-evident that the working with smaller amountsof solvents also results in a considerable economic improvement.

The application of dry powders containing the vitamins of the B groupmakes possible the use of percolators which are insofar advantageousthat on the one hand a removal of impurities through fractionalapplication of various solvents is easily possible and on the other handthe main portion of impurities remains behind undissolved.

Also, as compared to the known methods of extracting dry powderscontaining vitamins of the B group with other solvents such as alcohols,the process of they present invent-ion has the advantage that a muchpurer extract or percolate by far is obtained, since solvents such astrichloroethylene, benzol and the like, do not at all, or only in smallproportion are able to dissolve the impurities present, which aredissolved by alcohols. Thus, the present invention presents therefore aconsiderable advance.

It is necessary in order to remove the vitamins of the i3 group from theorganic phase and to carry it over into the aqueous phase, according tothe prior processes, always to utilize large amounts of liquids, usuallymore than the volume of the organic phase. It has now been found thatdisadvantages of the hitherto utilized processes can be overcome by theprocess of the present invention. This is based on the surprisingdiscovery that the vitamins of the B group can quite generally betransferred from the phenol-containing solvent or solvent mixture by theaddition of a small amount of certain oxygencontaining polar organicsubstances which displace the vitamins into the aqueous phase.

This will be illustrated by the following examples:

A concentrate is taken which is obtained from the running of apurification of the vitamins of the B group from a natural substrate. Ifthis concentrate is extracted by means of trichloroethylene plusp-chlorophenol, there is obtained for example cc. of extract whichbesides various impurities consists of 20% p-chlorophenol in trichloroethylene with 7.5-10 mg. of vitamin B To this extract is added 50cc. of Water. In the thus resulting two-phase system the vitamins of theB group are found exclusively in the organic phase, while the aqueousphase is practically colorless and contains practically no vitamins ofthe B group. This mixture is then mixed and shaken with one of thefollowing substances in the designated amount, which is alwayscalculated upon 100 cc. of the organic phase:

Ethylacetate 3.5 Ethyl ether 5.0 Dioxane 5 .0 Acetone 5 .0Methylethylketone 6.0 n-Butanol 7.5

Amylalcohol 7.5 Methanol 15.0

In each case, the vitamins of the B group go over very suddenly andpractically quantitatively into the aqueous phase. The action is veryrevealing and surprising. Since the remaining colored impurities in theorganic phase remain to a considerable degree, the aqueous solution hasan intensive and pure red color.

It is self-evident that the figures given above with respect to theamounts of the oxygen-containing substances are only guiding figures andmay be very greatly varied. The addition of a polar oxygen-containingorganic substance to the two-phase system of water/hydrocarbon plusphenolic substance shifts the distribution coefiicient of the vitaminsof the B group in favor of the water; in other words the additionincreases the solubility of the vitamins of the B group in water.According to whether more or less oxygen-containing polar substance isadded, the distribution coefficient is shifted to a greater or lesserextent, which again has the result that the connected extraction of thevitamins of the B group with Water can be carried out with more or lessfrequent extractions by shaking.

The importance of the process of the present invention for thepurification of vitamins of the B group is particularly evident if thepresent invention is compared with the known methods of proceeding. Ananalogous effect with regard to the transferring of the vita mins of theB group from the organic into the aqueous phase can only occur accordingto the known processes first by the addition of an equal volume oftrichloroethylene, chloroform, hydrocarbon, carbon disulfide, or asomewhat lesser volume of benzol and the like, in other words in theabove example only by the addition 'of at least 75-100 cc. of solvent.The difference is therefore extraordinarily apparent. While with theknown processes the utilized solvent only has the function of dilutingthe phenol-containing organic phase to such extent that the phenol losesits property of holding the vitamins of the B in the organic phase,according to the present invention there is no diluting action but theaddition of the oxygen-containing polar agents plays a decided role,giving a completely new effect, which results in a considerable savingof organic solvents.

The amounts given in the above table of polar oxygencontainingsubstances causes, in the absence of water, already the formation of afioculation containing the vitamins of the B group in the phenolorphenol-derivative containing solvents. A further addition of extractingagent can serve for complete precipitation of the vitamins of the Bgroup. This occurs for ketones, esters, ethers, and the like; in otherwords for each polar substance in which the vitamins of the B group arenot soluble. Alcohols produce in the above given amounts also afloculation, which however is not quantitative. If by the addition of asuitable oxygen-containing polar substance to a mixture of phenolor aphenol-derivative with a suitable solvent containing the vitamins of theB group a total or only partial lioculation of the vitamins of the B(ill group occurs, this is unimportant. In each case accord ing to theprocess of the present invention, the solubility of the vitamins of theB group in the concerned medium is lowered to such extent that withoutfurther treatment the vitamins can be extracted with water.

The theoretical explanation of the action of the above namedoxygen-containing polar substances is that these substances have theability of forming a solvate or complex with the phenol. Such solvate orcomplex of the phenol molecule loaded with the polar molecule no longerhas the property of serving as carrier for the vitamins of the B group.As a result of this, the phenol or phenol derivative no longer has theability of causing polar mediums which are not solvents for the vitaminsof the 13 group to have a solution effect for these vitamins. Neither dophenols have the capacity of increasing the solubility for vitamins ofthe B group in polar mediums which are capable of dissolving thesevitamins, in the event that the molecular ratio of phenol/polar mediumis less than 1. Polar media which contain abnormally high additions ofphenol have the capacity of dissolving the vitamins of the B group andto extract the same from water. This observation is in good agreementwith the ascertainment that for example very small amounts of methanol(i. e. 0.5%) in a solution of phenol and chloroform considerably alterthe absorption spectrum of the phenol. Even 2.5% of methanol in.chloroform acts so that the absorption spectrum of the phenol takes upthe same design as pure methanol. This means, therefore, that alreadywith this slight amount of methanol a complete solvating of the phenoltakes place.

It has now been found that the vitamins of the B group, according to thepresent invention, with very slight amounts of various phenolicsubstances can be caused to precipitate from the aqueous phase. Thehitherto needed concentrations of suitable phenols in the very diluteaqueous solution of the B group vitamins generally lay under thesaturation concentration of theparticular phenol in water. Only in veryimpure aqueous solutions, such as raw concentrates, are somewhat higherconcentrations of phenolic substances necessary for the quantitativeprecipitation of the vitamins of the B group.

This precipitation is tied up with a substantial purification of thevitamins of the B group. Substances which with phenols give noprecipitation are practically completely separated. To this group belongabove all the brown impurities which for example are present in theextracts of digested sludge.

The novel and truly unexpected discovery that the vitamins of the Bgroup can be precipitated from aqueous solutions by phenols, which havebeen previously confirmed as excellent solvents for these vitamins, isillustrated by the following:

It is known that phenols with oxygen compounds such as ethers,aldehydes, ketones, esters, acids, as Well as with amines and acidamides form complexes which are often very difficultly soluble. (P.Pfeilfer, Organische Molekiilverbindungen in Stereochemie, Leipzig undWein 1932.) By the addition of a slight amount of a suitable phenol tothe aqueous solution of the vitamins of the B group, the observedprecipitate is probably such a type of complex. The vitamin B moleculecontains sufiicient oxygen and nitrogen atoms to be able to serve aselectron donator for the formation of such a type of complex. Thecomplexes formed with the suitable phenolic substances of the vitaminsof the B group which without exception are difficulty soluble in waterdissolve excellently in an excess of the particular phenolic substance.They go therefore into the liquid phenol-rich phase or in the solidphenol phase it an excess of phenolic substance is utilized. If thephenolic substance is utilized not as such, but as a mixture with ahydrocarbon or with carbon disulfide, the vitamins of the B group asphenolic substance complexes go into the organic phase.

In the case of the precipitation with phenol as well as the extractionwith a phenol mixture from the aqueous solution, it is not the freevitamins of the B group which are dealt with'but their complex compoundswith phenols which are very well dissolved in the corresponding phenols,and the phenol-rich phase of the phenolwater-mixture, as well as in thephenolic substance-hydrocarbon-mixture and in the phenolicsubstance-carbon disulfide-rnixture, but not however in water or in thepoor in phenol, phenolic substance-water-phase.

For the formation of complexes of phenols with the vitamins of the Bgroup, the following conditions are valid, which can be determined fromthe figures of Table 2 below considering also the previously giveninformation:

(1) The complex formation occurs under conditions such that a reactionbetween the two partners is possible, for example in aqueous solution ofthe two constituents, or between the vitamin B group being in solutionand the solid phenolic substance or molten phenolic substance or asolution of the phenol, or between vitamins of the B group in solidstate and molten or dissolved phenolic substances. For practicalpurposes, it is necessary that the complex formation is favored to occurif the concerned phenol is present in molten state.

Solid phenols react, therefore, in general favored by temperatures whichA lie at or higher than their melting point. Many phenols form howevercomplexes also in the solid state. The following observations are basedupon the reactions of the phenols with aqueous solutions of vitamins ofthe B group at customary or higher temperatures. For practical purposesit is very valuable that the complex formation is practicallyquantitative and in the least concentration of the vitamins of the Bgroup and that in this manner an isolation of the vitamins of the Bgroup is possible from very dilute solutions.

(2) For the formation of the complex it is necessary that the phenoliccompound contains a free hydroxyl group. Etherated or esterified phenolsare therefore unsuitable.

(3) Phenolic substances With more than one hydroxyl group, such as diortri-hydroxy-benzols, are not suitable for complex formation.

(4) Phenolic compounds with oxygen containing substituents such asnitro, carbinol, and carbonyl groups and the like, and also suchcontaining amino groups have no or only an extremely weak ability toform complexes with the vitamins of the B group.

(5) Besides phenol itself, the numerous derivatives of themonohydroxybenzols as Well as the naphthols are suitable for theformation of complexes. This ability is influenced positively ornegatively by the type and position of the particular substitution to aregular extent, that is increased or decreased, and in many cases alsocompletely eliminated, as maybe seen from the following:

a. Non-oxygen-containing, negatively-charged substituents, such ashalogens fundamentally increase the binding ability of the phenol ifthey are in meta or para position. The meta position increases thebinding ability more than does the para position.

b. Substituents such as alkyl groups have practically no influence ifthey are in the meta or para position.

0. All substituents in the ortho position weaken the complex formingability of the phenol.

d. By more than one substituent the type and position thereof have aneven greater effect. While the placing in the meta and para positionsalso by more negatively charged radicals in general has a strengtheningaction, the occupying of the two ortho positions has a completelyundesirable influence on the complex forming ability. Thus phenolicsubstances in which the two ortho positions are substituted by alkyl orhalogen groups do not form any complex in the cold with the vitamins ofthe B group (in aqueous solution). In this case however 10 since twochlorines in the 'ortho position in a substituted phenol still form inmolten state, that is while hot, a complex by shaking with the aqueoussolution of the vitamin B group, but not however with the analagousdibromo compounds.

The following examples serve to clarify these observations andconditions:

The fact that ortho substituted phenols have less of a capability offorming complexes than the non-ortho substituted may be explained onlyon the basis of steric hindrance. The larger the ortho placedsubstituent, the slower reacting of the phenol hydroxyl group. Thelarger bromine atom hinders the reaction ability of the phenol hydroxylgroup to a greater extent than does the chlorine atom. Accordingly,2,4,6-tribromophenol is no longer able, and in fact also not even inmolten state, to bind the vitamins of the B group. The phenol which ischlorinated in the same manner however still has the ability of formingcomplexes, but only in the molten state. These observations correspondwith findings which were made in completely diiierent relation. Thus,for example, 2,6-di-tertiarybutyl-4-methylphenol, with most phenolreagents no longer shows the typical phenol reactions. The inability ofreaction of this compound can be traced back to the steric hindrance ofthe phenol hydroxyl group by the ortho position-found two tertiary butylgroups (N. C. K. I ones et al., Ind. Eng. Chem. 44, 2721 (1952) Forpractical carrying out of the precipitation of the vitamins of the Bgroup, the corresponding amount of phenolic substances is stirred in theaqueous solution of the vitamin and the stirring is continued for sometime. With solid phenols, a warming of the mixture is for the most partnecessary to a temperature at which the phenol is molten. According tothe solubility of the particular phenols in water there is formed eitherimmediately, or after several hours, a turbidity. After standing for ashort time the complex compound of the vitamins of the B group depositsa red oily or solid precipitate on the bottom 'which may subsequently beseparated.

To facilitate the separation of this precipitate from the aqueousliquid, preferably a porous carrier such as diatomaceous earth orcellulose powder, is stirred together with the precipitate subsequentlyresulting in a quick the molecular size of the substituents has aninfluence clearing of the liquid.

The obtained combined effect of the simultaneous ap-' plication ofphenolic substance and porous carrier is arousing the likelihood ofadsorption of the vitamins of the B group on the carrier, which afterthe precipitation is equally red colored. It has however been shown thatthese carriers do not act as adsorbents. In tests carried out withoutcarrier or with enormously high amounts of carrier, pactically the sameprecipitating effect can be obtained, as is shown in Table 2.

TABLE 2 Percent Phenol Percent Diatomaceous earth Thus, diatomaceousearth (or cellulose powder) plays TABLE 3 Precipitation ability of thevitamins of the B group from aqueous solution by various phenolsutilizing a vitamin B solution obtained out of digested sludge with acontent of 30 gamma vitamin B per each cc. with the addition of 1%diatomaceous earth as filter aid:

12 At pH values above 8.5, the precipitation ability is lowered. Theprecipitating capacity of the sediment and the clearing capacity of theliquid, particularly after the addition of diatomaceous earth orpowdered cellulose, increase with lowered pH values.

T he separation of the vitamin B group-containing sediment can be eitherthrough filtration or by the aid of a centrifuge.

The recovery of the vitamins of the B group from the separated andpossibly diatomaceous earth-or pom cellulose-containing or other filteraids-containing sediment can be carried out in the following ways:

(a) The sediment can be mixed, also in wet state, with a hydrocarbon orcarbon disulfide to which additional amounts of a phenol have beenadded, for extraction purposes, whereby the vitamins of the B group arePercent content of phenolic substances in the solution, necessary forcomplete Solubility of N0. Phenolic substance precipitation of B1z(A)phenol substances =100A in H2003) B in the cold in the hot (molten)henol 8.2 97.5 o-chlorophenoL- 2.8 78.5 o-bromophenol very slightlysoluble.

rn-chlorophenol p-chlorophenolp-bromophenoL. 2,5-dichlorophenol. 2.6dichloropheno1 2,4-dichlorophenol 2,4,6-trih10rophenol. noprecipitation-.- about 2.5.. 2,4,6-tribromophenol o no precipit o-cresol2.6 m-cresol 2.4.

p-cresol- (Schloro-E-cresoi 6-chloro-2-cresol 4-eh1oro-2-cresol.precipitation 5-chloro-2-creso1 o 2,5-dimethyl-l-oxybenzol.- noprecipitation..- 3,5-dimethyl-i-oxybenzol do 3,edimethyl-i-oxybenzol--p-tertiary-amylphenol... poxy-diphenyl o-oxy-diphenyl. thymol less than1.0..

much more than 1.0. -...-do

do practically no prccipitation. carvacrol.. precipitatiomprecipitation.mnaphthohno precipitation... about 2.

26 B-naphthol do more than 2-....

very slightly soluble. 0.075

The fact that in order to completely precipitate the vitamin B group,variable amounts of the various phenolic substances are necessary, ispartially explained by the above given conditions and partially in thatthe individual phenolic substances have different solubilities in water.In general, the need of phenols is less if the phenol is moredifiicultly soluble in water. There is found namely between water andthe vitamins of the B group a competition for the phenolic substance,whereby an equilibrium is set up as to the distribution of the phenolicsubstances between the vitamins of the 13 2 group and water, the ratioof the distribution being dependent upon the capacity of the phenols tocombine with the two competing substances.

The figure B of Table 3 characterizes approximately the ability of thephenolic substances to bind Water. The figure C tells to a certaindegree if the particular phenolic substance can bind the vitamins of theB group better or worse, compared to the water-binding ability. Thelower the figure C, the bettercompared with water--can the vitamins ofthe B group be bound by the phenolic substance.

The above conditions apply also to the aqueous solutions of crystallizedvitamins of the B group, and not only for the above given vitamin Bconcentrate. Very impure raw extracts need for complete precipitation ofthe vitamins of the B group somewhat more phenols,

some times as much as double the amount, as compared transferred intothe mixture of phenol plus hydrocarbon or carbon disulfide. In this casethe complexremains unchanged.

(b) The sediment can be stirred with a water-soluble substance, inwhich, however, the vitamins of the B group are not soluble, such as forexample with a lower ketone, and then filtered on? and dried. By thisprocedure, the complex is broken up and the phenol component removed.After mixing the thus obtained powders, forexample with Water, thevitamins of the B group g0 immediately into solution.

(0) The sediment can be stirred with an alcohol and filtered. Thevitamins of the B group appear together with the phenols in thefiltrate. In this case, the complex is likewise broken up.

In any method of proceeding according to a-c above, a furtherpurification of the vitamins of the B group occurs consisting of aremoval of proteins, colored substances and the like.

The following examples are given as illustrative only of the process ofthe present invention, the scope of the present invention not howeverbeing limited to the specific examples given.

Example 1 cc. of a red-brown colored vitamin B concentrate obtained outof digested sludge and which contains according to the microbiologicaltest with E.coli-mutant 113-3, 30 gamma of vitamin B per cc., in otherwords a total of 3 mg, is brought to a pH of 7.0 and is extracted with20, 10 and 10 cc. of a 20% solution of pp 13 chlorophenol intrichloroethylene. .The dark red extract is purified and washed oncewith 10 cc. of a sodium bicarbonate solution and subsequently threetimes, each time with cc. of water. The presently obtained extract of 40cc. can with advantage be utilized for further purification andobtaining of crystallized vitamin B Example 2 136cc. of an aqueousvitamin B concentrate obtained out of digested sludge and containing 40gamma of vitamin B per cc. is brought to a pH of 7.0 and mixed with 1.4g. of diatomaceous earth and 78 g. of ammonium sulfate. After dissolvingthe ammonium sulfate, a red-like fioccular sediment precipitates out andis filtered. The wet, red-colored filtered residue (about 8.3 g.) is bytriturating with 7.6 g. of water-free sodium sulfate transformed into adry powder. The thus obtained dry powder is subsequently finallypulverized in a mortar and mixed with 25 cc. of trichloroethylene. Thethus obtained slurry is put in a percolator. As soon as the surfacelevel of the liquid has almost reached the upper level, a mixture oftrichloroethylene with 5% p-chlorophenol is introduced. This mixtureextracts only insignificant amounts of the vitamins of the B group aswell as yellow-colored substances. Only after raising the p-chlorophenolconcentration in the trichloroethylene to 10% is about 35 cc. of a darkred colored percolate obtained. The further percolation with the likesolvent mixture yields an only weakly yellow-colored flow. The column iscolored brown after the percolation and contains the main portion ofimpurities. The percolate containing the vitamins of the B group servefor further purification of the vitamins of the B group until theircrystallization.

Example 3 50 cc. of concentrate obtained out of digested sludgecontaining the vitamins of the B group in an amount of 40 gamma per cc.is mixed with 29 g. of ammonium sulfate and subsequently extracted fourtimes, each time with 10 cc. of n-butanol. The butanol extract isevaporated to dryness in vacuum at a temperature of about 3040 C. Theobtained residue is extracted with 5 cc. of a mixture of l g. phenol and4 cc. of trichloroethylene, the extract subsequently washed four timeseach with 1 cc. of water and the thus purified extracts are if necessarywith advantage further worked up to crystallized vitamin B Example 4 50cc. of the same vitamin B concentrate as in Example 3 is stirred with 29g. of ammonium sulfate and 0.5 g. of diatomaceous earth. The flocculatedred sediment is filtered. This wet product of 3.0 g. quantity is mixedwith 2.8 g. of water-free sodium sulfate and triturated to a powder. Thedry powder obtained in this manner from the vitamin B group concentrateis subsequently stirred with acetone and a percolator is filledtherewith. After running through of the acetone there is an after-Washwith 5 cc. of trichloroethylene and it is then percolated with 15 cc. oftrichloroethylene containing 15% phenol. The remaining percolatematerial has a brown color and the total vitamin B activity is found inthe red colored percolate from which with advantage crystallized vitaminB can be obtained.

Example 5 40 cc. of a solution which contains 2.9 mg. of the vitamins ofthe B group along with various impurities in a solvent mixtureconsisting of p-chlorophenol and trichloroethylene and which has beenobtained by a purification process of the vitamins of the B group out ofdigested sludge, and wherein the dry substance is about 1% vitamins ofthe B group, is mixed with 2 cc. of n-butanol and shaken four times,each time with 10 cc. of water. The purified, intensively red-coloredaqueous extract is washed twice each with 10 cc. of n- 14 butanol, andthree times each with 15 cc. of ethyl ether. There is thus obtained apure red-colored aqueous solution which contains 2.8 mg. vitamins of theB group. The solid substance of this solution contains now the vitaminsof the B group in a concentration of about 6%.

Example 6 35 cc. of the same starting material as in Example 5 obtainedfrom a vitamin B concentrate, consisting of a mixture of 10%p-chlorophenol in trichloroethylene with a content of 5.2 mg. ofvitamins of the B group is mixed with 3.5 cc. of methylethyl ketonewhereby a carmine red floccular sediment percipitates. This isseparated, washed with trichloroethylene and subsequently dissolved in 5cc. of water. The obtained solution can with advantage be utilized forthe further purification and crystallization of the vitamins of the Bgroup.

Example 7 To 500 cc. of a purified aqueous solution at pH 3.5 containing25 gamma per cc. of the vitamins of the B group is added 11 g. ofp-chlorophenol and 5 g. of diatomaceous earth. After 10 minutes ofshaking there is obtained a floccular, easily separable sediment whichis filtered with the help of a glass suction filter G3, subse quentlystirred with 20 cc. of acetone, filtered, washed with acetone and freedof acetone in a vacuum dessicator. The red-colored powdered sediment isstirred with 20 cc. of water and filtered. The filtered residue iswashed with a small amount of water. The vitamins of the B group arenowfound quantitatively in the filtrate, whereby the concentration of thevitamins of the B group in the aqueous solution to about 20 times, aswell as a final substantial purification in a quick and careful manner,has occurred.

Example 8 To 200 cc. of a purified aqueous solution at pH 3.5 containing40 gamma per cc. of vitamins of the B group is mixed 16 g. of phenol and2 g. of diatomaceous earth. After shaking for a short time the floccularred sediment is filtered, washed with 8% aqueous phenol solution,subsequently stirred several times each with 10 cc. of a mixture of 20parts phenol and parts trichloroethylene and filtered. The red-coloredphenol-trichloroethyleneextract contains concentrated the vitamins ofthe B group in practically quantitative yield in substantially purestate and can be further Worked up in the usual manner.

Example 9 4.5 cc. of an eluate concentrate obtained out of digestedsludge which contains 70 gamma per cc. of the vitamins of the B groupalong with various impurities at pH 3.5 is shaken with 0.18 g.p-chlorophenol and 0.18 g. of diatomaceous earth for 10 minutes.Subsequently, the precipitated sediment is filtered and stirred with 5cc. of acetone. After filtering, washing with acetone and drying invacuum, the red colored powder is stirred with 1 cc. of water, filteredand washed with a small amount of water. The vitamins of the B group arenow found in the aqueous filtrate in practically pure form and inpractically quantitative yield.

Example 10 cc. of the same concentrate as in Example 1 is brought to apH of 7.0 and extracted four times with 100 cc. 'of a 15 solution ofm-chlorophenol in o-dichlorobenzol. The combined extracts are washedtwice, each time with 5 cc. of a 5% sodium bicarbonate solution, andsubsequently three times, each time with 5 cc. of water. The thusobtained extract may be utilized with advantage for further purificationand obtaining of crystallized vitamin B Example 11 100 cc. of the sameconcentrate as in Example 1 is brought to a pH of 6.5 and extracted fourtimes, each time with 10 cc. of a 15% solution of 6-chloro-3-cresol incarbon tetrachloride. The combined extracts are washed twice, each timewith 5 cc. of a 5% sodium bicarbonate solution and subsequently twotimes, each time with 5 cc. of water. In this manner there is obtainedan extract which is suitable for further purification and obtaining ofcrystallized vitamin B Without further analysis, the foregoing will sofully reveal the gist of the present invention that others can byapplying current knowledge readily adapt it for various applicationswithout omitting features which, from the standpoint of prior art,fairly constitute essential characteristics of the generic or specificaspects of this invention and, therefore, such adaptations should andare intended to be comprehended within the meaning and range ofequivalence of the following claims.

What is claimed as new and desired to be secured by Letters Patent is:

1. In a process of producing vitamins of the B group, the steps oftreating a concentrate containing vitamins of the B group with anorganic solvent mixture comprising a mono-halogenated phenol selectedfrom the group consisting of mono-halogenated phenols having the halogengroup in the position meta and mono-halogenated phenols having thehalogen group in the position para to the hydroxyl group of said phenol,dissolved in a liquid organic substance in which said vitamins of the Bgroup are insoluble and being selected from the group consisting ofhydrocarbons, halogenated hydrocarbons and carbon disulfide, therebyforming an extract solution of said vitamin of the B group dissolved insaid solvent mixture; treating the thus-formed organic extract solutioncontaining the vitamins of the B group with water and with a watersoluble alcohol, thereby causing due to said alcohol substantiallyquantitative transfer of said vitamins of the B group into the aqueousphase; saturating the thus-obtained aqueous solution containing saidvitamins of the B group dissolved therein with a phenol selected fromthe group consisting of meta and para chlorophenol in the presence of aninert solid porous absorbent material, thereby forming a precipitatecontaining the oily Waterinsoluble complex of said vitamins of the Bgroup and said phenol absorbed on said absorbent; and recovering thethus-precipitated vitamins of the B group.

2. In a process of producing vitamins of the B group, the steps oftreating a concentrate containing vitamins of the B group with anorganic solvent mixture comprising a mono-halogenated phenol selectedfrom the group consisting of mono-halogenated phenols having the halogengroup in the position meta and mono-halogenated phenols having thehalogen group in the position para to the hydroxyl group of said phenol,dissolved in a liquid organic substance in which said vitamins of the Bgroup are insoluble and being selected from the group consisting ofhydrocarbons, halogenated hydrocarbons and carbon disulfide, therebyforming an extract solution of said vitamins of the B group dissolved insaid solvent mixture;

treating the thus-formed organic extract solution containing thevitamins of the B group with Water and with a Water soluble alcohol inan amount sufficient only to cause precipitation of said vitamin B groupfrom said organic extract solution and thus cause dissolution of saidvitamins of the B group in said water, thereby causing due to saidalcohol substantially quantitative transfer of said vitamins of the Bgroup into the aqueous phase; saturating the thus-obtained aqueoussolution containing said vitamins of the B group dissolved therein witha phenol selected from the group consisting of meta and parachlorophenol in the presence of an inert solid porous absorbentmaterial, thereby forming a precipitate containing the oilywater-insoluble complex of said vitamins of the B group and said phenolabsorbed on said absorbent; and recovering the thus-precipitatedvitamins of the B group.

3. In a process of producing vitamins of the B group, the steps oftreating a concentrate containing vitamins of the B group with anorganic solvent mixture comprising a mono-halogenated phenol selectedfrom the group consisting of mono-halogenated phenols having the halogengroup in the position meta and mono-halogenated phenols having thehalogen group in the position para to the hydroxyl group of said phenol,dissolved in a liquid organic substance in which said vitamins of the Bgroup are insoluble and being selected from the group consisting ofhydrocarbons, halogenated hydrocarbons and carbon disulfide, therebyforming an extract solution of said vitamins of the B group dissolved insaid solvent mixture; treating the thus-formed organic extract solutioncontaining the vitamins of the B group with water and with a watersoluble alcohol, thereby causing due to said alcohol substantiallyquantitative transfer of said vitamins of the B group into the aqueousphase; saturating the thusobtained aqueous solution containing saidvitamins of the B group dissolved therein with a phenol selected fromthe group consisting of meta and para chlorophenol in the presence of aninert solid porous absorbent material, thereby forming a precipitatecontaining the oily waterinsoluble complex of said vitamins of the Bgroup and said phenol absorbed by said inert solid porous absorbentmaterial filtering said precipitate; washing the thus obtained filteredprecipitate with a low molecular weight ketone so as to free saidvitamins of the B group from said phenol; and recovering the thus freedvitamins of the B group.

References Cited in the file of this patent UNITED STATES PATENTS2,530,416 Wolf Nov. 21, 1950 2,613,171 Jackson Oct. 7, 1952 2,643,968McCormick June 30, 1953 2,678,900 Denkewalter May 18, 1954 FOREIGNPATENTS 675,247 Great Britain July 9, 1952

3. IN A PROCESS OF PRODUCING VITAMINS OF THE B12 GROUP, THE STEPS OFTREATING A CONCENTRATE CONTAINING VITAMINS OF THE B12 GROUP WITH ANORGANIC SOLVENT MIXTURE COMPRISING A MONO-HALOGENATED PHENOLS SELECTEDFFROM THE GROUP CONSISTING OF MONO-HALOGENATED PHENOLS HAVING THE HALOGENGROUP IN THE POSITION META AND MONO-HALOGENATED PHENOLS HAVING THEHALOGEN GROUP IN THE POSITION PARA TO THE HYDROXY GROUP OF SAID PHENOL,DISSOLVED IN A LIQUID ORGANIC SUBSTANCE IN WHICH SAID VITAMINS OF THEB12 GROUP ARE INSOLUBLE AND BEING SELECTED FROM THE GROUP CONSISTING OFHYDROCARBONS HALOGENATED HYDROCARBONS AND CARBON DISULFIDE, THEREBYFORMING AN EXTRACTSOLUTION OF SAID VITAMINS OF THE B12 GROUP DISSOLVEDIN SAID SOLVENT MIXTURE, TREATING THE THUS-FORMED ORGANIC EXTRACTSOLUTION CONTAINING THE VITAMINS OF THE B12 GROUP WITH WATER AND WITH AWATER SOLUBLE ALCOHOL, THEREBY CAUSING DUE TO SAID ALCOHOL SUBSTANTIALLYQUANTITITATIVE TRANSFER OF SAID VITAMINS OF THE B12 GROUP INTO THEAQUEOUS PHASE, SATURATING THE THUSOBTAINED AQUEOUS SOLUTION CONTAININGSAID VITAMINS OF THE B12, GROUP DISSOLVED THEREIN WITH A PHENOL SELECTEDFROM THE GROUP CONSISTING OF META AND PARA CHLOROPHENOL IN THE PRESENCEOF AN INERT SOLID POROUS ABSORBENT MATERIAL, THEREBY FORMING APRECIPITATE CONTAINING THE OILY WATERINSOLUBLE COMPLEX OF SAID VITAMINSOF THE B12 GROUP AND SAID PHENOL ABSORBED BY SAID INERT SOLID POROUSABSORBENT MATERIAL FILTERING SAID PRECIPITATE, WASHING THE THUS OBTAINEDFILTERED PRECIPITATE WITH A LOW MOLECULAR WEIGHT KETONE SO AS TO FREESAID VITAMINS O THE B12 GROUP FROM SAID PHENOL, AND RECOVERING THE THUSFREED VITAMINS OF THE B12 GROUP.